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3-Part Diff TechnoIogy

pocH-100i
Automated HaematoIogy AnaIyser
2 pocH-100i / Product Training July 2005
: Specifications
: Measurement principles
: Histogram interpretation
: Flagging
pocH-100i: 3-Part Diff TechnoIogy
pocH-100i / Product Training July 2005
pocH-100i - Specifications
pocH-100i / Product Training July 2005
Parameters
WhoIe bIood mode (WB): 19 parameters
WBC, RBC, HGB, HCT, MCV, MCH, MCHC, PLT
LYM%, MXD%, NEUT%, LYM#, MXD#, NEUT#,
RDW-SD, RDW-CV, PDW, MPV, P-LCR
+ WBC, RBC & PLT histograms
PrediIuted Mode (PD): 8 parametres
Throughput 1 seconds/sample
SampIe VoIume
·WhoIe bIood mode: 15 µL aspirated
1 mL sample required, 500 µL in case of micro tubes
·Pre-diIuted mode: 20 µL
(auto-dispense is available)
20 µL cap. blood with 1:26 dilution
Measurement
PrincipIes
Direct current (DC) detection method: WBC
Hydrodynamic focusing DC detection method: RBC/PLT
Non-cyanide HGB analysis: HGB
pocH-100i - Specifications
5 pocH-100i / Product Training July 2005
SampIe ID 15 digits
Printer Built-in thermal printer
Interface Host communication by TCP/ÌP (LAN) or serial port
Options Bar code reader
MuItiIanguage English, French, German, Ìtalian, Spanish, Japanese
Dimensions
(W x D x H (mm)
Weight (kg)
Main Unit: 15 x 60 x 50,
approx. 1
Data Storage 100 sample results with histograms
6 QC files. Each files can store up to 21 parameters by 60
points
pocH-100i - Specifications
6 pocH-100i / Product Training July 2005
WBC: DC detection method
RBC/PLT: Sheath flow DC detection method
HGB: Non-cyanide HGB detection method
HCT: Cumulative pulse height detection
method
: Measurement principles
pocH-100i - Specifications
pocH-100i / Product Training July 2005
: WBC: white blood cell count
: RBC: red blood cell count
: HGB: haemoglobin
: HCT: hematocrit (%RBC vol. to whole
blood vol.)
: PLT: platelet count
: MCV: mean corpuscular volume
(HCT/RBC)
: MCH: mean corpuscular hemoglobin
(HGB/RBC)
: MCHC: mean corpuscular hemoglobin
concentration
(HGB/HCT)
AnaIyticaI Parameters
pocH-100i / Product Training July 2005
: RDW-SD: RBC distribution width ÷
standard deviation
Anisocytosis (RBC)
Monitoring of blood transfusion
: RDW-CV: RBC distribution width ÷
coefficient of variation
Anemia of spherule shaped
: PDW: platelet distribution width
PLT agglutination
RBC overlap
AnaIyticaI Parameters
pocH-100i / Product Training July 2005
: MPV: mean platelet volume
Hematopoiesis function of PLT
Movement of PLT inside the body
: P-LCR: platelet large cell ratio
PLT agglutination
RBC overlap
Hematopoiesis function of PLT
AnaIyticaI Parameters
10 pocH-100i / Product Training July 2005
: SCR%: lymphocyte ratio (LYM%)
: MCR%: mixed leucocyte ratio (MXD%)
(monocyte, eosinophil, basophil)
: LCR%: neutrophil ratio (NEUT%)
: SCC#: lymphocyte count (LYM#)
: MCC#: mixed leucocytes count (MXD#)
: LCC#: neutrophil count (NEUT#)
AnaIyticaI Parameters
11 pocH-100i / Product Training July 2005
ReproducibiIity (WB)
: Reproducibility study for all parameters by 10
consecutive analyses in WB mode
: Low SD
: Low CV (%)
: For exact data please refer to scientific
documents (product file)
12 pocH-100i / Product Training July 2005
ReproducibiIity (PD)
: Reproducibility study for all parameters by 10
consecutive analyses in PD mode
: Low SD
: Low CV (%)
: For exact data please refer to scientific
documents (product file)
1 pocH-100i / Product Training July 2005
CorreIation with KX-21
: WB and PD: very good correlation with other instruments
(KX-21 or SE-500)



KX-21N vs pocH-100i WBC
0
20
0
60
0
100
120
10
0 20 0 60 0 100 120 10
KX-21N WBC X

p
o
c
H
-
1
0
0
i

W
B
C

X

1
0
^
9
/
L

KX-21N vs pocH-100i RBC
0
1
2


5
6


0 1 2 5 6
KX-21N RBC X

p
o
c
H
-
1
0
0
i

R
B
C

X

1
0
^
1
2
/
L

KX-21N vs pocH-100i HGB
0
2

6

10
12
1
16
1
20
0 2 6 10 12 1 16 1 20
KX-21N HGB g/dL
p
o
c
H
-
1
0
0
i

H
G
B


g
/
d
/
L

KX-21N vs pocH-100i HCT
10
15
20
25
0
5
0
5
50
55
60
65
10 15 20 25 0 5 0 5 50 55 60 65
KX-21N HCT %
p
o
c
H
-
1
0
0
i

H
C
T

%

KX-21N vs pocH-100i MCV
0
50
60
0
0
0
100
110
120
10
10
0 50 60 0 0 0 100 110 120 10 10
KX-21N MCV fL
p
o
c
H
-
1
0
0
i

M
C
V

f
L

KX-21N vs pocH-100i MCH
10
15
20
25
0
5
0
5
50
10 15 20 25 0 5 0 5 50
KX-21N MCH pg
p
o
c
H
-
1
0
0
i

M
C
H

p
g

KX-21N vs pocH-100i MCHC
20
25
0
5
0
5
20 25 0 5 0 5
KX-21N MCHC g/dL
p
o
c
H
-
1
0
0
i

M
C
H
C

g
/
d
L

KX-21N vs pocH-100i PLT
0
200
00
600
00
1000
1200
0 200 00 600 00 1000 1200
KX-21N PLT X 10^9/L
p
o
c
H
-
1
0
0
i

P
L
T
X

1
0
^
/
9
/
L

KX-21N vs pocH-100i W-SCC
0
25
50
5
100
125
0 25 50 5 100 125
KX-21N W-SCC
p
o
c
H
-
1
0
0
i

W
-
S
C
C

r
2
= 0,9968 r
2
= 0,9943 r
2
= 0,9945
r
2
= 0,9293 r
2
= 0,9923 r
2
= 0,9856
r
2
= 0,8737 r
2
= 0,9928 r
2
= 0,9973
1 pocH-100i / Product Training July 2005
Carryover
: Carryover study for CBC 5 parameters with control
blood (WBC, RBC, HGB, HCT, PLT)
: Procedure:
Sample is measured times consecutively
WB and PD
Carryover ratio = (B1-B) / (S-B) x 100 (%)
B1: 1st measurement of background count
B: rd measurement of background count
S: rd measurement of sample
: Results: no carryover
: For exact data please refer to scientific documents
(product file)
15 pocH-100i / Product Training July 2005
HaemogIobin Detection with pocH-100i
16 pocH-100i / Product Training July 2005
HaemogIobin
: Haemoglobin is the red blood dye
: Chemically, haemoglobin is an iron-
containing protein
: Ìt is produced in the erythroblasts of the bone
marrow
: Haemoglobin is present in the erythrocytes
: The haemoglobin concentration is directly
linked to the HCT and the RBC concentration
: Haemoglobin is responsible for transport of
oxygen and CO
2
1 pocH-100i / Product Training July 2005
New borns 1 - 26 g/dl
Children 10 - 26 g/dl
Women 12 - 16 g/dl
Men 1 - 1 g/dl

-
r
r-
.
.-
~ .·. :.. ^...··. .. .
HaemogIobin - Reference Ranges
Conversion factors: g/dl mmol/L x 0.6206
mmol/L · r..rr
1 pocH-100i / Product Training July 2005
: Erythrocytes are lysed in a dilution of 1:500
: 1. Reaction step:
HBG with the bound Fe
2+
is oxidised by the Potassium
hexocyanoferrate (K

(Fe(CN)
6
) to become
Methemoglobin (with Fe
+
).
: 2. Reaction step:
Methemoglobin reacts with Potassium cyanide (KCN),
building the red and stable Cyan hemoglobin-complex
with a maximum in absorption at 56 nm.
: The extinction at the maximum of absorption is directly
proportional to the Hemoglobin content of the whole
blood
HaemogIobin Reference Method
(DIN 58931)
1 pocH-100i / Product Training July 2005
HaemogIobin Detection History
: Ìdentical measurement principle as the reference
method.
: Measurement in the photometer under the same
conditions.
: Also the reaction conditions are identical.
: Cyanide containing reagent for haemolysis.
Semi automated systems = Quicklyser
Fully automated systems= Stromatolyser-C
: Disposal of cyanide containing waste needs a
permission.
20 pocH-100i / Product Training July 2005
: pocH-pack L (ÌÌ) is used for the haemoglobin
and the leukocyte analysis
: pocH-pack L(ÌÌ) contains quaternary
ammonium salts (MTAC and LTAC)
: Very good correlations between the Sysmex
method and the reference method
HaemogIobin - Cyanide-free Method
21 pocH-100i / Product Training July 2005
1. Lysis of RBC by quaternary ammonium salts
2. Change of conformity
. Oxidation of Fe
2+
at the haeme group
(Fe
2+
Fe
+
)
HaemogIobin - Cyanide-free Method
22 pocH-100i / Product Training July 2005
Haemoglobin molecule
RBC
Ammonium
salts
. .
Fe
2+
-
Fe
2+
-
RBC
. .
-
-
Fe
2+
Fe
2+
1. Lysis of RBC
Fe
2+
Fe
2+
Fe
2+
Fe
2+
2 pocH-100i / Product Training July 2005
Haemoglobin molecule
RBC
. .
Fe
2+
-
Fe
2+
-
RBC
. .
- -
Fe
2+
Fe
2+
2. Change of Conformity
Fe
2+
Fe
2+
Fe
2+
Fe
2+
2 pocH-100i / Product Training July 2005
Haemoglobin molecule
Methemoglobin-complex
. .
- -
Fe
+
Fe
+
. .
Fe
2+
-
Fe
2+
-
O
2
3. Oxidation
Fe
2+
Fe
2+
Fe
2+
Fe
2+
25 pocH-100i / Product Training July 2005
Wavelength (nm) 50 60
A
b
s
o
r
p
t
i
o
n
$5··.. ' ¬.....
´5¬-5. ´··`
2
max
= 555 nm
HemogIobin - SpectraI AnaIysis
26 pocH-100i / Product Training July 2005
: photometrical analysis at 555 nm
HGB Photometer
lens
Sample stream Cellpack
Flow cell photosensor LED
2 pocH-100i / Product Training July 2005
Start
HGB-chamber is rinsed with diluent
HGB-blank is determined and stored
Measurement of the sample
in a dilution of 1:500
Sample - Blank = HGB-result
Printing of the result
End
HGB Measurement - FIow Chart
2 pocH-100i / Product Training July 2005
HGB Measurement
Mixing Chamber
Transducer Chamber
HGB Flow Cell
2 pocH-100i / Product Training July 2005
: Haematocrit is the ratio (%) of total volume of
RBCs to whole blood volume
: Determination by centrifugation or calculation
: Besides haemoglobin, hematocrit is a measure for
anaemia
Haematocrit (HCT)
0 pocH-100i / Product Training July 2005
New borns 0,51 - 0,65
Children 0,5 - 0,
Women 0, - 0,
Men 0,2 - 0,52
0
0,1
0,2
0,3
0,4
0,5
New borns Babies Children Women Men
Hematocrit - Reference Ranges
1 pocH-100i / Product Training July 2005
: Volume determination of the corpuscular particles
in the blood
: Normally, equivalent to the pure erythrocyte mass
: Blood is aspirated into special capillaries, which
contain dried heparin
: The capillaries are sealed and put into a dedicated
centrifuge
: Ìn the high-turning centrifuge the non-coagulating
blood is separated into its solid and fluid parts
using the different specific weights of plasma and
blood particles
Hematocrit Centrifugation
2 pocH-100i / Product Training July 2005
Hematocrit Centrifugation
Total volume V
T
Centrifuge
V
T
V
HCT (%) = (V / V
T
) x100
pocH-100i / Product Training July 2005
0
0,1
0,2
0,
0,
0,5
0,6
0,
0,
0,
1,0
Leukocytes
(Buffy Coat)
Erythrocytes
: The reading is done on a scale, which is based
on the principle of similar triangles.
: The guide, which cuts through the
border between erythrocyte-
and leukocyte-layer gives
the Hematocrit-value.
Hematocrit Centrifugation
pocH-100i / Product Training July 2005
Transducer
Start-Sensor
Stop-Sensor
Defined volume V
T
Hematocrit - CumuIative PuIse Height
Detection Method
5 pocH-100i / Product Training July 2005
Ph = k x V
Ery
Ph pulse height
k constant
V
ery
volume of one RBC
V
Ery
= 1 / k x Ph
Ph
Hematocrit - CumuIative PuIse Height
Detection Method
6 pocH-100i / Product Training July 2005
V
T
V
HCT (%) = (V / V
T
) x 100
V = V
Ery
= (1 / k) Ph
HCT (%) = (1 / V
T
k) Ph x 100
Ph Pulse height
k Constant
V
Ery
Volume of one RBC
Ph = k x V
Ery
V
Ery
= (1 / k) x Ph
Hematocrit - CumuIative PuIse Height
Detection Method
V
T
Total volume
V Volume of all RBCs
pocH-100i / Product Training July 2005
Transducer
chamber
Ph
HCT (%) = V / V
T
x 100
HCT (%) = (1 / V
T
k) Ph x 100
V
V
T
Hematocrit - CumuIative PuIse Height
Detection Method
Start-Sensor
Stop-Sensor
Defined volume V
T
pocH-100i / Product Training July 2005
Mean Cell Volume (MCV)
HCT (%)
RBC (x 10
6
/µl)
MCV (fl) =
Mean Cellular Hemoglobin (MCH)
HBG (g/dl)
RBC (x 10
6
/µl)
MCH (pg) =
Mean Cellular Hemoglobin Concentration (MCHC)
HBG (g/dl)
HCT (%)
MCHC (g/dl) =
RBC Quotients: RBC, HGB, HCT
pocH-100i / Product Training July 2005
100
Deviation from median in %
-0
-20
0
20
0
60
0
. ~ -rr . .'.·. ·. ..- ´ · ·.- ´
·.. '·. . . ¬... ·.....·.. · .·.
.···.... .. ~.'. · ·.. .·.···
WBC
10

/µl
5,0
10
PLT
10

/µl
1
HGB
g/dl
,5
RBC
x 10
6
/µl
2
HCT
%
0
MCV
fl

MCHC
g/dl
1
MCH
pg
BioI. Variation of CBC Parameters
0 pocH-100i / Product Training July 2005
SampIe Processing - WB
: Blood aspiration: 15µL
: Dilution ratio:
RBC/PLT: approx.165 times
WBC/HGB: approx. 500 times
: 1st dilution:
15µL blood + 1.5mL diluent (10 times)
: 2nd dilution:
RBC/PLT: 200µL 1st sample + 1.mL diluent
(10.5 times; 10 x 10.5 = 165)
WBC/HGB: 61µL 1st sample + 20µLdiluent + 50µL lyse
(.5 times; 10 x .5 = 500)
1 pocH-100i / Product Training July 2005
SampIe Processing - PD
: Blood collection: 20µL
: Dilution ratio:
RBC/PLT: approx. 20 times
WBC/HGB: approx. 1000 times
: Pre-dilution:
20µL blood + 500µL diluent (26 times)
: 1st dilution:
15µL PD sample + 1.55mL diluent (260 times)
: 2nd dilution:
RBC/PLT: 200µL 1st sample + 1.mL diluent (10.5
times; 260 x 10.5 = 20)
WBC/HGB: 61µL 1st sample + 20µL diluent + 50µL
lyse
(.5 times; 260 x .5 = 1000)
2 pocH-100i / Product Training July 2005
DC Detection Method
·. 5.. · ..··.
pocH-100i / Product Training July 2005
vacuum
blood cells
resistance
direct current
(approx. 100 V)
internal electrode external electrode
aperture
sample beaker
sample
PrincipIe DC Detection Method
pocH-100i / Product Training July 2005
external
electrode
internal
electrode
aperture
vacuum
U = R x Ì
DC Detection Method
5 pocH-100i / Product Training July 2005
Samples are passing through the centre of the
aperture with sheath flow solution
Accurate RBC/PLT count
Axial cell flow
Recirculation and coincidence are prevented
Sheath reagent: pocH-pack D (ÌÌ)
Hydrodynamic focusing system: RBC/PLT
Hydrodynamic Focusing
6 pocH-100i / Product Training July 2005
Sample can be more concentrated with HDF:
: Dilution
KX-21N: RBC/PLT: 25.000 times (WB und PD)
pocH-100i: RBC/PLT: 165 times (WB); 20
times (PD)
Hydrodynamic focusing system: RBC/PLT
Hydrodynamic Focusing
pocH-100i / Product Training July 2005
DC Detection Method
: Absolute counting
Defined volume of the diluted blood sample is
brought into the RBC/PLT detector line for
measurement
Valves separate this defined volume
Electric stepping motor drives the syringe, which
pumps the defined volume into the detector unit
pocH-100i / Product Training July 2005
DC Detection Method
Syringe Unit No.21
(diluent)
Syringe Unit No. 22
(sample and sheath)
pocH-100i / Product Training July 2005
aperture
sample
defined
volume
vacuum
start
stop
counter
off
display:
S puIse / voIume
on
pulse management digital
discriminator
signal-pulses
noise-pulses
signal-pulses
Not to be changed by the user:
sample volume;
dilution ratio;
analysed volume;
diameter of the orifice
The instrument automatically checks: counting
time, condition of the orifice during the analysis
(noise-control)
Advantages:
no calibration of the counts
DC Detection Method
~...· ..·..
50 pocH-100i / Product Training July 2005
Counting of particles in a
defined volume
Counting of particles per time
unit
AbsoIute Counting ReIative Counting
Cell concentration is
determined by a known
sample volume and a known
dilution
The counting rate is determined
indirectly by a reference
sample. A similar behaviour of
sample and calibrator - resulting
in comparable results - is
assumed.
No calibration of the counting
results needed
Calibration of the counting
results is mandatory.
DC Detection Methods - Comparison
51 pocH-100i / Product Training July 2005
r . - - . · r rr r. r r-
·
.
.

5.. ...·
·. 5.. · ..··.. 5.. ·.·.
DC Detection Method
52 pocH-100i / Product Training July 2005
r
.

r . - - . · rrr r.rr-
r . - - . · rrr r.rr-
t
i
m
e
puIse height
CumuIative
Distribution Curve
PuIse Diagram
(0 cells)
- r r . - - - . r
c
e
I
I
s
DC Detection Method
5 pocH-100i / Product Training July 2005
r . - - . · rrr r.rr-
Histogram
r
.

r . - - . · rrr r.rr-
CumuIative
Distribution Curve
- r r . - - . r -
c
e
I
I
s
DC Detection Method
5 pocH-100i / Product Training July 2005
CumuIative Distribution Curve
Histogram
0
500
1000
1500
2000
2500
000
0 6 12 1 24 0 36 2 48 5 60 66 72 84 0 96 10210811120
Maximum
·. 5.. · ..··.
DC Detection Method - Histogram
55 pocH-100i / Product Training July 2005
.--- '. .-.- '.
rythrocyte (RBC) Histogram
: RBC size: 0-100 fL
: RBC detection: between 25 and 250 fL
: Distribution curves are separated by flexible
discriminators
RL RU
RBC
PLT
56 pocH-100i / Product Training July 2005
.-. '. r.- '.
'.· ·
r. '.
PIateIet (PLT) Histogram
PL PU
PLT
RBC


: PLT size: -12 fL
: PLT detection: between 2 and 0 fL
: Fixed discriminator at 12 fL
5 pocH-100i / Product Training July 2005
Measurement FIow Chart
Detector
WBC
ChanneI
HGB
ChanneI
RBC/PLT
ChanneI
WhoIe
BIood
SampIe
Information
WBC
Scattergram
RBC
Scattergram
PLT
Scattergram
Method
DC Detection
Method
Non-cyanide
HGB Method
HDF DC
Detection Method
Parameters
WBC
LYM# LYM%
MXD# MXD%
N&T# N&T%
HGB
MCV
MCH
MCHC
RBC
HCT
RDW-SD
RDW-CV
PLT
PDW
MPV
P-LCR
5 pocH-100i / Product Training July 2005
Summary: PrincipIes and
TechnoIogies pocH-100i
: RBC / PLT / WBC: DC Detection Method
: HGB: cyanide-free photometric analysis at 555 nm
: HCT: cumulative pulse height detection method
: Histograms: RBC, PLT, WBC
: Histogram flagging: suspect flags
: RBC parameters: RBC, HGB, HCT, MCV, MCH,
MCHC, RDW-SD, RDW-CV
: PLT parameters: PLT, PDW, MPV, P-LCR
: WBC parameters: WBC, W-SCR, W-MCR, W-LCR, W-
SCC, W-MCC, W-LCC
5 pocH-100i / Product Training July 2005
PrincipIes & TechnoIogies
nd of Part I
Time for questions.
60 pocH-100i / Product Training July 2005
Histogram Interpretation
^:^ - 5·.··
Leukocyte histogram
Lymphocytes in % and absolute
Eo, Mono, Baso in % and absolute
Neutrophiles in % and absolute
rythrocyte histogram
Erythrocyte distribution width
Thrombocyte histogram
Thrombocyte distribution width
Mean Platelet Volume
Platelets - Large Cell Ratio
WBC
RBC
PLT
61 pocH-100i / Product Training July 2005
CBC: Reference Ranges
RBC Men .6-6.2 x 10
6
/µl x 10
12
/l
Women .2-5. x 10
6
/µl x 10
12
/l
HGB Men 1-1 g/dl ,5-11,0 mmol/l
Women 12-16 g/dl ,5-10,0 mmol/l
HCT Men - % 0,-0, mmol/l
Women 6-6 % 0,6-0,6 mmol/l
MCV 5-5 fl
MCH 2- pg 1,6-2,05 fmol
MCHC 2-6 g/dl 1,-22, mmol/l
RDW-SD -6 fl (width in 20% peak height)
RDW-CV 11-16 % (calculated from width in 60 % peak height)
Parameter Sex Convent.&nits SI-&nits
62 pocH-100i / Product Training July 2005
DispIay of AnaIysis ResuIts
AnaIysis data without preceding sign are within preset Iimits
If anaIysis error has occurred and a vaIue is not avaiIabIe,
one of the foIIowing is dispIayed
Sign xposition
! Value is out of the linearity limit
+ Result exceeds the upper patient limit
- Result exceeds the lower patient limit
* Result is unreliable
Sign xposition
"+++.+¨ Value exceeds display range.
"***.*¨ Value could not be calculated because of analysis error. At this time,
the analysis error flag. {Error] (inverse in coloration display) appears.
"---.-¨ Value could not be calculated due to data error, or volume distribution
analysis parameter is not displayed when the analysis was performed
in pre-diluted mode
6 pocH-100i / Product Training July 2005
Histogram error fIags
FIag xposition
RL Error at the lower discriminator for RBC
R& Error at the upper discriminator for RBC
DW Distribution width (20%) can be not calculated
MP There are multiple peaks
PL Error at the lower discriminator for PLT
P& Error at the upper discriminator for PLT
AG The particle count equal to or less than WBC-LD has exceed the range
WL Error at the lower discriminator for WBC
W& Error at the upper discriminator for WBC
T1 Trough discriminator T1 could not be determined
T2 Trough discriminator T2 could not be determined
F1 Small cells are inaccurate
F2 Middle cells are inaccurate
F3 Large cells are inaccurate
6 pocH-100i / Product Training July 2005
Curve does not start at the base line.
flag " RL ", abnormal height at the lower discriminator
PossibIe cause:
· Large PLT
· Fragmented RBC
· PLT aggregation
CAUTÌON:
All results flagged with " RL " should be checked!!
LD
RBC
PLT
RBC Histogram FIagging
65 pocH-100i / Product Training July 2005
Curve does not end at the baseline
UD
RBC
flag " RU ", abnormal height at the upper discriminator
UD
RBC
PossibIe cause:
· Cold Agglutinins
· Erythroblasts
Normoblasts
CAUTÌON:
The RBC-result and all results flagged with " RU "
should be checked!!
RBC Histogram FIagging
66 pocH-100i / Product Training July 2005
RBC Histogram FIagging
flag " MP ", Multiple peaks
RBC RBC
PossibIe cause:
: Transfusion: patient's and donor's RBCs have different size
: Unlysed and aged sample
6 pocH-100i / Product Training July 2005
flag "DW ", Distribution width
RBC RBC
: Ìt is impossible to determine the distribution width,
because the histogram does not cut the 20%-line for a
second time.
: This curve is only an example and might also show a
different course.
RBC Histogram FIagging
6 pocH-100i / Product Training July 2005
flag "DW ", Distribution width
RBC RBC
: Various size of cells are present
: Review by manual method
RBC Histogram FIagging
6 pocH-100i / Product Training July 2005
.-$.. - -. '.
r ´
. ´
'`
.-^ ´´` ~ r ´r-.` ´r÷.`
r ´
r .
u
.·... ·..·
.... ´
' .. ..
'`
RBC Histogram Distribution Width
0 pocH-100i / Product Training July 2005
PLT 150-50 x 10³/µl x 10

/l
PDW -1 fl (Width in 20% peak height)
MPV -1 fl
P-LCR 15-5 %
Parameter Convent.&nits SI-&nits
PLT Histogram: Reference Ranges
1 pocH-100i / Product Training July 2005
PLT Histogram
Curve is marked off by two discriminators
:The distribution curve should lie between the discriminators and
start and end at the base line
:The measurement area is between 2 and 0 fl
2 pocH-100i / Product Training July 2005
PLT Histogram FIagging
: PLT, Platelet count
: Parameter from the thrombocyte-histogram
MPV, Mean Platelet Volume between the
upper and the lower discriminator
Normal range: - 12 fl
Use for control of erythropoiesis
P-LCR, Platelet - Large Cell Ratio
Normal range: 15 - 5 %
Elevation might be hint for:
Thrombocytes aggregates
Micro erythrocytes
PDW, Platelet Distribution Width in 20 %
of total curve height
Normal range: - 1 fl
Elevation might be hint for:
Thrombocytes aggregates
Micro erythrocytes
Erythrocyte fragments
Pct (%)
PLT (x 10

/µl)
MPV (fl) =
12 fl
LD UD
PLT
P-LCR
100 %
20 %
PDW
pocH-100i / Product Training July 2005
PLT Histogram FIagging
Curve does not start at the base line.
flag " PL ", abnormal height of the curve at the lower
discriminator
PLT PLT
PossibIe cause:
· Blank is too high
· Cell fragments or cell ruins
CAUTÌON:
PLT-result and all results flagged with " PL "
should be checked!
Thrombocytes also in the counting chamber,
if needed!
pocH-100i / Product Training July 2005
PLT Histogram FIagging
flag " PU ", abnormal height of the curve at the upper discriminator
PossibIe causes:
: Coagulation of blood
: Pseudo-thrombocytosis
: Fragmented RBC
: Large platelets
: Thrombocyte aggregates
CAUTÌON:
PLT-result and results flagged with " PU "
should be checked!
Thrombocytes: counting chamber
Curve does not end at the base line
5 pocH-100i / Product Training July 2005
PLT Histogram FIagging
flag " MP ", Multiple peaks
PossibIe cause:
: Transfusions, the patient's and the donor's PLTs have
different sizes
: Pathological cells: CML (chronic myeloic leukemia)
6 pocH-100i / Product Training July 2005
PLT Histogram FIagging
flag " DW ", Distribution width
PLT
:The distribution curve should lie between the
discriminators and start and end at the base line
:The measurement area is between 2 and 0 fl
pocH-100i / Product Training July 2005
PLT Histogram FIagging
flag " DW ", Distribution width
PLT
: Microcytes or fragmented RBC
: PLT size dissimilitude and cryoglobulins
pocH-100i / Product Training July 2005
WBC grown ups -10 x 10

/µl x 10

/l
children up to 12 x 10

/µl x 10

/l
babies up to 15 x 10

/µl x 10

/l
W-SCR grown ups 25-0 %
children, babies up to 0 %
W-MCR grown ups -1 %
W-LCR grown ups 50-0 %
W-SCC grown ups 1- x 10

/µl x 10

/l
children up to 5 x 10

/µl x 10

/l
babies up to 6 x 10

/µl x 10

/l
W-MCC grown ups 0,2-1 x 10

/µl x 10

/l
W-LCC grown ups 2- x 10

/µl x 10

/l
Parameter Age range Convent.&nits SI-&nits
Reference Ranges
pocH-100i / Product Training July 2005
Schematic picture
of a leukocyte
Mitochondrion
Nucleus
Nucleolus
Cell membrane
Ribosome
Cytoplasm
Electrolyte-solution
After lysis
WBC Histogram: InfIuence of the
Iysing reagent
0 pocH-100i / Product Training July 2005
WBC Histogram: InfIuence of the
Iysing reagent
:'· ..
0 2 6 10 12 1 16 1 20 22
Neutrophiles
Basophiles
Eosinophiles
Monocytes
Lymphocytes
Cell diameter in µm
10 - 15
- 1
11 - 16
12 - 20
- 12
0 - 0
60 - 120
0 - 10
0 - 10
120 - 250
Cell volume in fL
Lymphocytes
Monocytes
Basophiles
Eosinophiles
Neutrophiles
~'·· ..
0 50 100 150 200 250 00
Lymphocytes
Monocytes
Basophiles
Eosinophiles
Neutrophiles
1 pocH-100i / Product Training July 2005
· Distribution curve should lie
within the discriminators (start
and end at base line)
· Lower discriminator (LD) is
flexible, but cannot be set below
0 fl
· Ìn the WBC channel WBC and
PLT are present (RBC are lysed)
· PLT volume is normally between
and 12 fl: LD separates WBC
from PLT and PLT are not
counted
Curve is marked off by 2 discriminators
0 50 100 150 200 250 00
UD ( fixed)
T2 T1 LD
WBC Histogram
2 pocH-100i / Product Training July 2005
flag " WL ", curve does not start at the base line
PossibIe causes:
: PLT aggregates or large PLT
: RBC lyse resistance
: Normoblasts (NRBC)
: Cold Agglutinins
CA&TION:
WBC - results and all results
flagged "WL¨ should be checked
WBC Histogram FIagging
pocH-100i / Product Training July 2005
WBC Histogram FIagging
flag " WU ", curve does not end at the base line
CA&TION:
Review by manual method or washing of
blood cells
Sample dilution? (high leukocytes value?)
: Ìmmature WBC
: Agglutination of WBC
pocH-100i / Product Training July 2005
T1 and T2 are so called trough discriminators:separation of WBC into
populations (F1, F2, F)
flags "T1¨ and "T2¨
0 50 100 150 200 250 00
UD ( fixed)
LD
T2 T1
population 1 = F1 population 2 = F2
population = F
F = fraction
· Discriminators are flexible in certain areas to adapt to the individual
sample
· Ìn abnormal cases the troughs are not able to separate between the
populations properly
WBC Histogram FIagging
5 pocH-100i / Product Training July 2005
WBC Histogram FIagging
Ìt is not possible in this
case to set T1 since there
was no valley found:
T1 is fIagged
flags "T1¨ and "T2¨
CAUTÌON:
· Ìf the sample is flagged
with T1 or T2, the results
from the pre-differential
should not be reported
· Manual method
· The WBC result is
reportable (if there is no
other flag). All leukocytes
are counted.
: Pathological cells (CML)
: Numerous abnormal blood cells
6 pocH-100i / Product Training July 2005
WBC Histogram FIagging
T1 was found, but no
second valley for T2:
T2 is fIagged
CAUTÌON:
· Ìf the sample is flagged
with T1 or T2, the results
from the pre-differential
should not be reported.
· The WBC result is
reportable (if there is no
other flag). All leukocytes
are counted.
: Numerous abnormal blood cells
: Aged sample
: Pathological cells (CML)
pocH-100i / Product Training July 2005
WBC Histogram FIagging
:All leukocytes are counted: total number of WBC is
correct (if no other flags are present)
:Valleys were found: T1 and T2 are not flagged
:But valleys are far from the base line (F1, F2, F)
flags "F1¨ , "F2¨ and "F¨
r .
pocH-100i / Product Training July 2005
WBC Histogram FIagging
:Monocytosis
:Eosinophilia
:Basophilia
:Unlysed
:Aged sample
flags "F1¨ , "F2¨ and "F¨
r .
CAUTÌON:
· Review by manual
method
· The WBC result is
reportable (if there is no
other flag). All leukocytes
are counted.
pocH-100i / Product Training July 2005
Histogram Interpretation: Cases (1)
Neutrophilia
Band
Seg
Lymph
Mono
Eo
Baso
%
%
%
%
1 %
0 %
Differential
WBC
LYM%
MXD%
NEUT%
Results
+ 2. x 10

/L
.1%
.%
.0%
(x 00)
WBC-Histogram
:^..... ·.... Neutrophilia
:Prominent peak with broad
distribution (NEUT%) for large
leukocytes.
:Ìn case of Lymphocytopenia a similar
curve is obtained.
0 pocH-100i / Product Training July 2005
Histogram Interpretation: Cases (2)
Lymphocytosis
Band
Seg
Lymph
Mono
Eo
Baso
Aty-Lym
%
20 %
6 %
%
5 %
0 %
%
Differential
WBC
LYM%
MXD%
NEUT%
Results
. x 10

/L
+ 6.%
15.%
÷ 1.5%
(x 1000)
WBC-Histogram
:^..... ·.... Lymphocytosis
:High, pointed peak in lympho area
(LYM%).
:Ìn case of Neutropenia a similar curve
is obtained.
1 pocH-100i / Product Training July 2005
Histogram Interpretation: Cases (3)
WBC-Histogram
PLT-Histogram
Case 1
Results
WBC
LYM%
MXD%
NEUT%
6.0 x10

/L
2.5%
.%
6.%
Results
PLT
PDW
MPV
P-LCR
+
+
6 x10

/L
1.6fl
12.fl
.%
(x 00)
The smear clearly shows that
platelets are aggregating. The
WBC histogram shows a peak in
the ghost area
( ) , while the PLT histogram
shows a wide distribution.
Although these large particles
usually affect the leukocyte counts,
the leukocytes distribution is well
separated from the ghost area on
the WBC histogram, probably
without any effect of small particles
in the ghost area. There is no WL
Alarm given .
2 pocH-100i / Product Training July 2005
Histogram Interpretation: Cases (4)
Case 2
WBC-Histogram Results
WBC
LYM%
MXD%
NEUT%
WL*
WL*
WL*
WL*
6. x10

/L
1.%
1.0%
.6%
Results
PLT
PDW
MPV
P-LCR
PU
DW
DW
DW
55 x10

/L
---.-fl
---.-fl
-.---%
PLT-Histogram
(x 00)
This sample contains larger
aggregation clusters as shown in
the smear. These clusters are
considered to affect the leukocyte
counts, because the distribution
curve on the WBC histogram
intersects the discriminator line
between the ghost and the Small
cell ratio at a high point, and the WL
flags are given. The PLT histogram
suggests the presence of large
particles. Analysis of a fresh blood
sample is required to obtain correct
platelet values.
pocH-100i / Product Training July 2005
Histogram Interpretation: Cases (5)
Ìnsufficient Lysing of Erythrocytes
WBC
LYM%
MXD%
NEUT
%
WL*
WL
WL
WL
. x10

/L
-.---
-.---
-.---
WBC-Histogram Results
(x 1000)
^. Iyse resistance RBC
· Histogram shows a pattern
typically seen in insufficient lysing
of RBC
· On the WBC histogram the
distribution curve intersects the
WBC lower discrimination line at
an abnormally high point. The WL
flag is output and asterisk marks
are put to the WBC value,
warning of low reliability of the
data
· This is frequently seen with
blood samples taken from
hepatic disease patients or very
early newborns. These problems
are solved by diluting the sample
or replacing plasma with cellpack
(blood cell washing)
pocH-100i / Product Training July 2005
Summary: PrincipIes & TechnoIogies
: RBC / PLT / WBC: DC Detection Method (HDF)
: HGB: cyanide-free photometric analysis at 555 nm
: HCT: cumulative pulse height detection method
: Histograms: RBC, PLT, WBC
: Histogram flagging: numerical flags and suspect flags
: RBC parameters: RBC, HGB, HCT, MCV, MCH,
MCHC, RDW-SD, RDW-CV
: PLT parameters: PLT, PDW, MPV, P-LCR
: WBC parameters: WBC, W-SCR, W-MCR, W-LCR, W-
SCC, W-MCC, W-LCC
5 pocH-100i / Product Training July 2005
Thank you for your kind attention!
Questions ?!

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